Gene transfer to eukaryotic cells has both in vivo and in vitro uses. As is well known, in vivo gene transfer to eukaryotic cells can be used to immunize a host, for therapeutic gene transfer to a host, and to study the biology of transferred genes in vivo. In vitro gene transfer to eukaryotic cells can be used to study simple and complex biological phenomena such as protein function, protein half-life, and gene-protein interactions. One preferred method for transferring genes to eukaryotic cells has been through the use of recombinant eukaryotic viruses. Although researchers and clinicians have enjoyed the many advantages of recombinant eukaryotic viruses for gene transfer to eukaryotic cells, the difficulty of constructing these viruses has impeded the rate at which new and useful gene transfer experiments and protocols have been developed.
Because of their large size, many recombinant eukaryotic viruses are produced via homologous recombination. Conventionally, homologous recombination used to generate large viral vectors has taken place in a host eukaryotic cell permissive for the growth of the recombinant virus (see, e.g., Berkner, BioTechniques, 6, 616–628 (1988)). Homologous recombination in eukaryotic cells, however, has at least two major drawbacks. The process is time consuming, and many preferred recombinant eukaryotic viral constructions are at a selective disadvantage relative to the predecessor eukaryotic viruses from which they are obtained. Therefore, if a skilled artisan attempts to create a new recombinant virus through the lengthy process of homologous recombination in a eukaryotic cell and fails to create the desired virus, that artisan is often unable to readily distinguish between the need to modify the construction technique and the possibility that the desired virus vector is not viable in the host cell. Accordingly, there is a need for new methods of generating eukaryotic gene transfer vectors.
Previous improvements in the generation of gene transfer vectors have included the use of yeast-based systems (Ketner et al., PNAS USA, 91, 6186–6190 (1994)), plasmid-based systems (Chartier et al., J. Virol., 70, 4805–4810 (1996); Crouzet et al., WO 96/25506), and cosmid-based systems (Miyake et al., PNAS USA, 93, 1320–1324 (1996)). While these systems can expedite the production of new recombinant eukaryotic viruses, additional flexibility and selection pressures are desired. The present invention provides a rapid and flexible method for producing new vectors, which can be used in gene transfer to eukaryotic cells in vitro and in vivo. The present invention also provides vectors modified for use in eukaryotic gene transfer, as well as methods and systems for using the same. These and other advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.